The development of the heart is controlled by transcription factor genes that govern the spatiotemporal patterns of expression of diverse downstream target genes. Accumulating evidence indicates that the homeobox class of transcription factor genes are of particular importance in regulating mammalian heart morphogenesis. To better understand the genetic programs of heart and lung development, four transcription factor genes=Pmx, S-8, Gsh-4, and lgl-have been cloned, chromosomally mapped, structures and expression patterns determined, and for three, a functional analysis was initiated by gene targeting. Gsh-4 mutants display respiratory control center defects resulting in early postnatal death, while S-8 mutants suffer early postnatal mortality, ventricular hypertrophy, and have cardiac contraction and relaxation rate abnormalities. It is proposed to conduct in depth physiological characterization of these mutant mice to better define the developmental roles of these genes. Moreover, as the S-8 and Pmx genes are extremely closely related in sequence and expression patterns, double Pmx-S-8 knockout mice will be made to explore the possibility of genetic redundancy. The next vertical advance in our understanding of the mechanisms by which these genes control heart and lung development will come from the identification nd characterization of their downstream genetic targets. To this end, it is proposed to use a battery of techniques, including PCR analysis of DNA binding sequences, differential display, and immunoprecipitation of target genes from chromatin. These experiments begin to define the genetic regulatory network of heart and lung development, and how genetic or epigenetic perturbations can result in congenital defects.